The overall goal of this project is the molecular characterization of the pH 6 antigen virulence determinant and the establishment of its role in the intracellular survival of Yersinia pestis KIM in macrophages. Dideoxy sequencing of a 1.7 kb region of Y. pestis DNA previously shown by others to contain the pH 6 antigen structural gene, psaA, and a regulatory locus, psaE, will be performed to identify open reading frames within these two loci. More importantly, analysis of the DNA in this region may identify the temperature and pH responsive sequences that mediate the optimal expression of this virulence determinant in an environment similar to that found inside a macrophage, that being 37 degrees C at pH 6. Genetic complementation of PsaA- and PsaE- mutants of Y. pestis using a plasmid vector carrying the intact loci will confirm the exclusive involvement of this sequence of Y. pestis chromosomal DNA in expression of the pH 6 antigen. The PsaA- and PsaE- mutants, as well as the parent PsaA+ PsaE+ Y. pestis KIM5 strain, will be used to infect resident murine peritoneal macrophages in culture. Both bacteriological culture and double immunofluorescent techniques will be used to enumerate intracellular bacterial survival to determine if the pH 6 antigen is involved in Y. pestis survival in macrophages. Elucidation of the mechanisms Y. pestis uses to detect environmental stimuli and to survive intracellularly will broaden our understanding of the pathogenesis of bubonic plague and may provide clues as to how other intracellular pathogens of man and animals cause disease.